Uridine

引言 糖基化 (Glycosylation) 是生物分子最普遍、最重要的修饰之一，这些修饰在细胞识别、信号传导、蛋白质折叠和稳定性以及免疫反应等生物过程中发挥关键作用【1】。已知的糖基化生物分子主要包括蛋白质和脂质。在一次偶然的实验中，斯坦福大学诺贝尔化学奖得主Carolyn Bertozzi的博士后Ryan Flynn发现，唾液酸的非天然分子SiaNAz (叠氮化唾液酸) 竟然可以通过细胞自身的代谢途径标记RNA分子。后续通过一系列的研究，题为Small RNAs are modified with N-glycans and displayed on the surface of living cells的论文于2021年在Cell上正式发表，这是科学家们首次意识到糖基化RNA存在的可能【2】。在此之后，关于RNA糖基化的标记、监测及生物功能的研究逐渐在各大期刊上发表。然而，很多科学家对糖RNA分子的存在存疑，其中一个至关重要的信息一直未被解析：RNA如何与N-聚糖连接在一起？这一信息对于验证糖RNA分子的真实存在以及后续研究糖RNA分子的合成途径和生物功能都不可或缺。然而，由于糖RNA分子含量极低（预计在总体RNA分子中的含量低于0.001%），依赖传统手段获取这一关键信息极其困难。 2024年8月21日，现为波士顿儿童医院助理教授的Ryan Flynn联合来自华盛顿圣路易斯大学医学院的生物质谱专家Benjamin Garcia课题组在Cell上发表了文章The modified RNA base acp3U is an attachment site for N-glycans in glycoRNA，首次证明了修饰的RNA碱基acp3U（3-(3-amino-3-carboxypropyl)uridine）是糖RNA中N-聚糖的结合位点，谢一轩博士（Garcia课题组）和柴培远博士（Flynn课题组）为论文共同第一作者【3】。 首先，为了更高效地标记糖RNA分子，研究团队开发了一种新型化学工具—rPAL。该技术在温和高碘酸氧化条件下 (7.5mM NaIO4, 10 分钟，室温)，与糖RNA上唾液酸中的邻二醇发生特异性反应。这一反应导致连接羟基的两个碳原子间的碳-碳键发生断裂形成醛基，再利用带有生物素的羟胺实现糖RNA分子的特异性标记。结果证明，rPAL技术不仅能够特异性标记糖RNA，而且与之前的SiaNAz方法相比，信号增益提高了150倍之多，大幅提升了对糖RNA的标记能力。 建立rPAL技术后，作者通过生物素-亲和素系统和强洗涤条件对糖RNA进行了富集，并对富集得到的糖RNA进行灵敏的RNA修饰质谱分析。结果显示，含有羧基的acp3U核苷在四种不同条件下均被广泛检测到。基于PNGase F酶释放反应通常会将会将N-糖与底物连接的酰胺转化为羧基，因此作者推测acp3U分子中的羧基很可能是由PNGase F酶解所产生。在含有氧16和氧18的水中分别对富集的糖RNA进行PNGase F释放后进行产物分析，结果显示acp3U在两个条件下分别产生了对应的氧16和氧18标记的质量 (346.12 m/z以及348.12 m/z)，并且其与acp3U标准样品在液相色谱中的保留时间及二级质谱信息完全吻合，这一结果证明了之前的猜想。进一步，研究团队通过糖苷内切酶Endo F2和 Endo F3对富集产物进行酶解，留下了单N-乙酰葡萄糖胺记号在RNA分子上，再通过靶向质谱鉴定出带有N-乙酰葡萄糖胺的acp3U化合物，为RNA与糖质相连提供了直接的证据。 模式图（Credit: Cell） 最后，研究团队在细胞中敲除了acp3U合成酶家族中的DWDT2，结果显示糖RNA信号显著降低，这一发现从生物学层面揭示了acp3U生物合成对糖RNA的影响，进一步证明了acp3U与糖RNA之间的联系。 总之，该研究首次揭示了糖RNA在分子结构和生物学基础方面的信息，为糖RNA的存在提供了直接证据，并为后续RNA糖基化的生物合成以及生物功能研究奠定了基础。值得一提的是，Ryan Flynn课题组近期再次联合Benjamin Garcia 课题组以及Daniel Bojar课题组在预印本bioRxiv发表了题为O-glycosylation contributes to mammalian glycoRNA biogenesis的文章，提供了RNA O-糖基化存在的证据【4】。这些发现不仅丰富了我们对RNA糖基化的理解，还为未来开发基于糖RNA的新型诊断和治疗策略提供了更多可能性。 参考文献 [1] Varki et al., Essentials of Glycobiology (4th edition), 2022. DOI: 10.1101/9781621824213 [2] Flynn et al., Cell, 2021. DOI: 10.1016/j.cell.2021.04.023 [3] Xie et al., Cell, 2024. DOI: 10.1016/j.cell.2024.07.044 [4] Porat et al., bioRxiv, 2024. DOI: 10.1101/2024.08.28.610074 https://www-cell-com.libproxy1.nus.edu.sg/cell/abstract/S0092-8674(24)00838-9 责编|探索君 排版|探索君 文章来源|“BioArt” End 往期精选 围观 一文读透细胞死亡(Cell Death) | 24年Cell重磅综述（长文收藏版） 热文 Cell | 是什么决定了细胞的大小？ 热文 Nature | 2024年值得关注的七项技术 热文 Nature | 自身免疫性疾病能被治愈吗？科学家们终于看到了希望 热文 CRISPR技术进化史 | 24年Cell综述

EnginZyme and Ajinomoto Bio-Pharma Services have developed a patented process to manufacture pseudouridine at industrial scale using biocatalysis in a sustainable, low-waste, patented process carried out in a cGMP* facility EnginZyme is in talks to help alleviate supply chain difficulties in low- and middle-income countries 2023 Nobel laureates discovered how pseudouridine could make mRNA suitable for vaccines STOCKHOLM--(BUSINESS WIRE)-- EnginZyme AB, a deep-tech company whose cell-free biomanufacturing technology uses the power of enzymes to create sustainable products and processes for a variety of industries, announced that it had patented a process to synthesize pseudouridine, a key ingredient in mRNA COVID-19 vaccines. EnginZyme and its CDMO partner, Ajinomoto Bio-Pharma Services, specialized in scale up and cGMP* manufacturing of small molecules and other high-value fine chemicals, said they could provide pseudouridine at a discount to buyers taking part in efforts to bolster the global supply chain of vaccine ingredients. The companies said they had already synthesized enough pseudouridine for more than half a billion doses of vaccine in a facility that is fully cGMP* compliant. The enzymatic process EnginZyme has perfected is cleaner and more efficient than the chemical synthesis methods currently used. A critical impurity, alpha-pseudouridine, which is a by-product of the chemical synthesis of beta-pseudouridine, is eliminated in EnginZyme's patented enzymatic synthesis. For more technical information, see our website. The 2023 Nobel Prize for Physiology or Medicine was awarded to Katalin Karikó and Drew Weissman, the pair who discovered that changing a chemical building block of messenger RNA – substituting pseudouridine for uridine – eliminated an inflammatory side effect that stalled development of vaccines based on mRNA. They made this discovery more than 15 years before the COVID-19 pandemic. During the COVID-19 crisis, the World Health Organization found that low- and middle-income countries were suffering from a lack of vaccine doses amid production and supply constraints, hoarding by wealthy countries, and prioritization of sales to governments that could pay the highest prices. "Not long after we discovered the process for making pseudouridine, we realized that we could produce a pure version at scale for much less than it was going for on the market," said Karim Engelmark Cassimjee, CEO of EnginZyme. "We decided the right thing to do would be to use this discovery to help make the global healthcare supply chain more resilient." He added that academic and nonprofit organizations could apply to receive free samples for research purposes. Geert Schelkens, R&D Manager at Ajinomoto Bio-Pharma Services, said: "The scale-up of a biocatalytic process and a green workup results in a drastic reduction of the compound's footprint. This achievement nicely aligns with our sustainability ambitions. This project with EnginZyme has been particularly rewarding because of its potential to improve the global supply chain for vaccine ingredients.” N1-methylpseudouridine-5’-triphosphate, which is derived from pseudouridine, helps stabilize and reduce the immunogenicity of mRNA. Messenger RNA technology made headlines through the successful COVID-19 vaccines produced by Pfizer-BioNTech and Moderna, and since then, an increasing number of early-stage therapeutics and emerging vaccines have been based on the technology. "Synthesizing pseudouridine with enzymes is so much more efficient than the chemical synthesis that is common today," said Matthew Thompson, head of enzyme development and innovation at EnginZyme. "It's a perfect example of what we are striving for: zero waste, less energy, and a cleaner result. We want to apply this across the spectrum of chemical manufacturing." *Current Good Manufacturing Practice regulations, enforced by the U.S. FDA, provide for systems that assure proper design, monitoring, and control of manufacturing processes and facilities. About EnginZyme EnginZyme is changing chemistry for good. We are enabling the shift to biomanufacturing by unlocking the power of enzymes for more cost-efficient and sustainable production of everyday products. Our patented enzyme immobilisation technology enables biomanufacturing without living organisms. It can be deployed using the same tools and techniques used in traditional chemical manufacturing, with lower costs and a much smaller environmental footprint. Based in Stockholm, EnginZyme is a growing company with a multidisciplinary team of experts in biocatalysis, organic chemistry, enzyme and process engineering, as well as AI and machine learning. This elite team is uniquely positioned to develop complete processes from techno-economic analysis all the way to industrial manufacture, incorporating a high degree of automation for safe, rapid, and reliable development. EnginZyme has been recognised as a Technology pioneer by the World Economic Forum and was selected to the Global Cleantech 100 list in 2022 and 2023. About Ajinomoto Bio-Pharma Services Ajinomoto Bio-Pharma Services is a fully integrated contract development and manufacturing organization with sites in Belgium, United States, Japan, and India, providing comprehensive development, cGMP manufacturing, and aseptic fill finish services for small and large molecule APIs and intermediates. Ajinomoto Bio-Pharma Services offers a broad range of innovative platforms and capabilities for pre-clinical and pilot programs to commercial quantities, including high potency APIs (HPAPI), biocatalysis, continuous flow manufacturing, Corynex® protein expression technology, oligonucleotide synthesis, antibody drug conjugations (ADC), and more. Ajinomoto Bio-Pharma Services is dedicated to providing a high level of quality and service to meet our client’s needs. Learn more: View source version on businesswire.com: Contacts Business development Karl Johan Tärbe karljohan@enginzyme.com Media Bogert-Magnier Communications James Connell jim@bogert-magnier.com +33 6 2152 1955 Elodie Doan Van elodie@bogert-magnier.com +33 6 8099 8881 Source: EnginZyme AB View this news release online at: